臺灣博碩士論文加值系統

實驗目的
研究NF-κB相關之microRNA以及細胞激素在大鼠之實驗性自體免疫前葡萄膜炎(EAAU) 之動態表現，並提出可能的免疫機轉分析。
實驗方法
本實驗以melanin-associated antigen在Lewis rat上誘發EAAU，並記錄臨床發炎程度以及前房液中白血球之數量。在進行虹膜、睫狀體、以及淋巴結之RNA萃取後，作者分析8個miRNA (miR-155-5p，miR-146a-5p，miR-182-5p，miR-183-5p，miR-147b，miR-21-5p，miR-9-3p，miR-223-3p)以及六個細胞激素(IFN-γ, IL-17, IL-12p35, IL-1β, IL-6, IL-10) 在病程中之動態變化。另外，為了確認上述實驗結果，也施作miRNA原位雜合以及ELISA以定量細胞激素。
結果
臨床發炎程度以及前房液中白血球之數量皆在疾病誘發後第15天達到高峰。miRNA分析發現miR-146a-5p、miR-155-5p、miR-223-3p、miR-147b在疾病中減弱表現，而miR-182-5p、miR-183-5p、及miR-9-3p則增強表現。細胞激素中，IFN-γ、IL-17、IL-12p35、IL-1β、及IL-6表現量上升，而IL-10表現則下降。
結論
miRNA的動態變化可能啟動NF-κB訊息傳遞，進而引發下游的Th17和Th1免疫細胞以及相關細胞激素之活化，以造成實驗性自體免疫前葡萄膜炎。

Purpose
To determine the dynamic changes of NF-κB-related microRNAs and cytokines in the course of experimental autoimmune anterior uveitis (EAAU) and elucidate the possible immunopathogenesis.
Materials and methods
Uveitis was induced in Lewis rats with bovine melanin-associated antigen. The clinical scoring of the inflammatory activity of the anterior chamber and leukocyte quantification in aqueous humor was done. RNA extraction from iris/ciliary body, and popliteal lymph nodes was performed to reveal the dynamic changes of 8 target miRNAs (miR-155-5p, miR-146a-5p, miR-182-5p, miR-183-5p, miR-147b, miR-21-5p, miR-9-3p, and miR-223-3p) and mRNAs of 6 cytokines (IFN-γ, IL-17, IL-12p35, IL-1β, IL-6, IL-10). miRNA in situ hybridization and ELISA quantification of cytokines were performed to confirm the results.
Results
The disease activity and leukocyte quantification was maximal on day 15 following immunization. miRNA profiling revealed down-regulation of miR-146a-5p, miR-155-5p, miR-223-3p and miR-147b, and up-regulation of miR-182-5p, miR-183-5p, and miR-9-3p. Cytokines analysis disclosed IFN-γ, IL-17, IL-12p35, IL-1β, and IL-6 over-expression with IL-10 down-regulation.
Conclusion
Dynamic changes of miRNAs were present in the EAAU course. By initiating NF-κB signaling, expressions of downstream cytokines and effector cells of Th17 and Th1 lineage were sequentially activated, and contributed to the disease.

口試委員會審定書………………………………………………………………..… i
序言及謝辭……………………………………………………………………….…. ii
中文摘要……………………………………………………………………………..iii
英文摘要……………………………………………………………………………..iv
緒論 (Introduction)…………………………………………………………………...1
研究方法與材料 (Materials and Methods) ………………………………………….4
結果 (Results) ………………………………………………………………………..9
討論 (Discussion) ………………………………………………………………...…12
參考文獻……………………………………………………………………………..20
表1…………………………………………………………………………….……..32
表2…………………………………………………………………………….……..33
圖1…………………………………………………………………………….……..34
圖2……………………………………………………………………………….…..35
圖3…………………………………………………………………………….….….36
圖4…………………………………………………………………………….….….37
圖5…………………………………………………………………………….….….38
圖6…………………………………………………………………………….….….39
圖7…………………………………………………………………………….….….41
圖8…………………………………………………………………………….….….42
附錄………………………………………………………………………….….……43

Amadi-Obi, A., C. R. Yu, X. Liu, R. M. Mahdi, G. L. Clarke, R. B. Nussenblatt, I. Gery, Y. S. Lee and C. E. Egwuagu. TH17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1. Nat Med. 2007. 13, 711-718.
Ambros, V. The functions of animal microRNAs. Nature. 2004. 431, 350-355.
Bandyopadhyay, S., M. E. Long and L. A. Allen. Differential expression of microRNAs in Francisella tularensis-infected human macrophages: miR-155-dependent downregulation of MyD88 inhibits the inflammatory response. PLoS One. 2014. 9, e109525.
Barker, B. R., D. J. Taxman and J. P. Ting. Cross-regulation between the IL-1beta/IL-18 processing inflammasome and other inflammatory cytokines. Curr Opin Immunol. 2011. 23, 591-597.
Boldin, M. P. and D. Baltimore. MicroRNAs, new effectors and regulators of NF-kappaB. Immunol Rev. 2012. 246, 205-220.
Boldin, M. P., K. D. Taganov, D. S. Rao, L. Yang, J. L. Zhao, M. Kalwani, Y. Garcia-Flores, M. Luong, A. Devrekanli, J. Xu, G. Sun, J. Tay, P. S. Linsley and D. Baltimore. miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice. J Exp Med. 2011. 208, 1189-1201.
Broekhuyse, R. M., E. D. Kuhlmann and H. J. Winkens. Experimental autoimmune anterior uveitis (EAAU). II. Dose-dependent induction and adoptive transfer using a melanin-bound antigen of the retinal pigment epithelium. Exp Eye Res. 1992. 55, 401-411.
Broekhuyse, R. M., E. D. Kuhlmann and H. J. Winkens. Experimental autoimmune anterior uveitis (EAAU): induction by melanin antigen and suppression by various treatments. Pigment Cell Res. 1993. 6, 1-6.
Broekhuyse, R. M., E. D. Kuhlmann, H. J. Winkens and A. H. Van Vugt. Experimental autoimmune anterior uveitis (EAAU), a new form of experimental uveitis. I. Induction by a detergent-insoluble, intrinsic protein fraction of the retinal pigment epithelium. Exp Eye Res. 1991. 52, 465-474.
Busskamp, V., J. Krol, D. Nelidova, J. Daum, T. Szikra, B. Tsuda, J. Juttner, K. Farrow, B. G. Scherf, C. P. Alvarez, C. Genoud, V. Sothilingam, N. Tanimoto, M. Stadler, M. Seeliger, M. Stoffel, W. Filipowicz and B. Roska. miRNAs 182 and 183 are necessary to maintain adult cone photoreceptor outer segments and visual function. Neuron. 2014. 83, 586-600.
Caso, F., L. Costa, D. Rigante, O. M. Lucherini, P. Caso, V. Bascherini, B. Frediani, R. Cimaz, E. Marrani, L. Nieves-Martin, M. Atteno, C. G. Raffaele, G. Tarantino, M. Galeazzi, L. Punzi and L. Cantarini. Biological treatments in Behcet''s disease: beyond anti-TNF therapy. Mediators Inflamm. 2014. 2014, 107421.
Caspi, R. R., C. C. Chan, B. G. Grubbs, P. B. Silver, B. Wiggert, C. F. Parsa, S. Bahmanyar, A. Billiau and H. Heremans. Endogenous systemic IFN-gamma has a protective role against ocular autoimmunity in mice. J Immunol. 1994. 152, 890-899.
Ceribelli, A., M. Satoh and E. K. Chan. MicroRNAs and autoimmunity. Curr Opin Immunol. 2012. 24, 686-691.
Chan, S. H., M. Kobayashi, D. Santoli, B. Perussia and G. Trinchieri. Mechanisms of IFN-gamma induction by natural killer cell stimulatory factor (NKSF/IL-12). Role of transcription and mRNA stability in the synergistic interaction between NKSF and IL-2. J Immunol. 1992. 148, 92-98.
Chen, Z. and J. J. O''Shea. Th17 cells: a new fate for differentiating helper T cells. Immunol Res. 2008. 41, 87-102.
Chi, W., X. Zhu, P. Yang, X. Liu, X. Lin, H. Zhou, X. Huang and A. Kijlstra. Upregulated IL-23 and IL-17 in Behcet patients with active uveitis. Invest Ophthalmol Vis Sci. 2008. 49, 3058-3064.
Dandona, L., R. Dandona, R. K. John, C. A. McCarty and G. N. Rao. Population based assessment of uveitis in an urban population in southern India. Br J Ophthalmol. 2000. 84, 706-709.
Durrani, O. M., C. A. Meads and P. I. Murray. Uveitis: a potentially blinding disease. Ophthalmologica. 2004. 218, 223-236.
Fang, I. M., C. P. Lin, C. M. Yang, M. S. Chen and C. H. Yang. Expression of CX3C chemokine, fractalkine, and its receptor CX3CR1 in experimental autoimmune anterior uveitis. Mol Vis. 2005. 11, 443-451.
Fang, I. M., C. H. Yang, C. P. Lin, C. M. Yang and M. S. Chen. Expression of chemokine and receptors in Lewis rats with experimental autoimmune anterior uveitis. Exp Eye Res. 2004. 78, 1043-1055.
Fang, I. M., C. H. Yang, C. P. Lin, C. M. Yang and M. S. Chen. Effects of pyrrolidine dithiocarbamate, an NF-kappaB inhibitor, on cytokine expression and ocular inflammation in experimental autoimmune anterior uveitis. J Ocul Pharmacol Ther. 2005. 21, 95-106.
Fang, I. M., C. H. Yang and C. M. Yang. Chitosan oligosaccharides attenuate ocular inflammation in rats with experimental autoimmune anterior uveitis. Mediators Inflamm. 2014. 2014, 827847.
Forrester, J. V., I. P. Klaska, T. Yu and L. Kuffova. Uveitis in mouse and man. Int Rev Immunol. 2013. 32, 76-96.
Fukunaga, K., T. Ishigami and T. Kawano. Transcriptional regulation of neuronal genes and its effect on neural functions: expression and function of forkhead transcription factors in neurons. J Pharmacol Sci. 2005. 98, 205-211.
Gritz, D. C. and I. G. Wong. Incidence and prevalence of uveitis in Northern California; the Northern California Epidemiology of Uveitis Study. Ophthalmology. 2004. 111, 491-500; discussion 500.
Gu, C., L. Wu and X. Li. IL-17 family: cytokines, receptors and signaling. Cytokine. 2013. 64, 477-485.
Hamzaoui, K., A. Hamzaoui, F. Guemira, M. Bessioud, M. Hamza and K. Ayed. Cytokine profile in Behcet''s disease patients. Relationship with disease activity. Scand J Rheumatol. 2002. 31, 205-210.
Ishida, W., K. Fukuda, T. Higuchi, M. Kajisako, S. Sakamoto and A. Fukushima. Dynamic changes of microRNAs in the eye during the development of experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci. 2011. 52, 611-617.
Jabs, D. A., R. B. Nussenblatt, J. T. Rosenbaum and G. Standardization of Uveitis Nomenclature Working. Standardization of uveitis nomenclature for reporting clinical data. Results of the First International Workshop. Am J Ophthalmol. 2005. 140, 509-516.
Kaneko, Y., G. S. Wu, S. Saraswathy, D. V. Vasconcelos-Santos and N. A. Rao. Immunopathologic processes in sympathetic ophthalmia as signified by microRNA profiling. Invest Ophthalmol Vis Sci. 2012. 53, 4197-4204.
Kelada, S., P. Sethupathy, I. S. Okoye, E. Kistasis, S. Czieso, S. D. White, D. Chou, C. Martens, S. M. Ricklefs, K. Virtaneva, D. E. Sturdevant, S. F. Porcella, Y. Belkaid, T. A. Wynn and M. S. Wilson. miR-182 and miR-10a are key regulators of Treg specialisation and stability during Schistosome and Leishmania-associated inflammation. PLoS Pathog. 2013. 9, e1003451.
Kurowska-Stolarska, M., S. Alivernini, L. E. Ballantine, D. L. Asquith, N. L. Millar, D. S. Gilchrist, J. Reilly, M. Ierna, A. R. Fraser, B. Stolarski, C. McSharry, A. J. Hueber, D. Baxter, J. Hunter, S. Gay, F. Y. Liew and I. B. McInnes. MicroRNA-155 as a proinflammatory regulator in clinical and experimental arthritis. Proc Natl Acad Sci U S A. 2011. 108, 11193-11198.
Lasiglie, D., E. Traggiai, S. Federici, M. Alessio, A. Buoncompagni, A. Accogli, S. Chiesa, F. Penco, A. Martini and M. Gattorno. Role of IL-1 beta in the development of human T(H)17 cells: lesson from NLPR3 mutated patients. PLoS One. 2011. 6, e20014.
Lopalco, G., L. Cantarini, A. Vitale, F. Iannone, M. G. Anelli, L. Andreozzi, G. Lapadula, M. Galeazzi and D. Rigante. Interleukin-1 as a common denominator from autoinflammatory to autoimmune disorders: premises, perils, and perspectives. Mediators Inflamm. 2015. 2015, 194864.
Lu, L. F. and A. Liston. MicroRNA in the immune system, microRNA as an immune system. Immunology. 2009. 127, 291-298.
Lu, T. X. and M. E. Rothenberg. Diagnostic, functional, and therapeutic roles of microRNA in allergic diseases. J Allergy Clin Immunol. 2013. 132, 3-13; quiz 14.
Luger, D., P. B. Silver, J. Tang, D. Cua, Z. Chen, Y. Iwakura, E. P. Bowman, N. M. Sgambellone, C. C. Chan and R. R. Caspi. Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category. J Exp Med. 2008. 205, 799-810.
Mauermann, N., J. Burian, C. von Garnier, S. Dirnhofer, D. Germano, C. Schuett, M. Tamm, R. Bingisser, U. Eriksson and L. Hunziker. Interferon-gamma regulates idiopathic pneumonia syndrome, a Th17+CD4+ T-cell-mediated graft-versus-host disease. Am J Respir Crit Care Med. 2008. 178, 379-388.
Morlon, A., A. Munnich and A. Smahi. TAB2, TRAF6 and TAK1 are involved in NF-kappaB activation induced by the TNF-receptor, Edar and its adaptator Edaradd. Hum Mol Genet. 2005. 14, 3751-3757.
Nussenblatt, R. B. The natural history of uveitis. Int Ophthalmol. 1990. 14, 303-308.
O''Connell, R. M., D. Kahn, W. S. Gibson, J. L. Round, R. L. Scholz, A. A. Chaudhuri, M. E. Kahn, D. S. Rao and D. Baltimore. MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity. 2010. 33, 607-619.
Rodriguez, A., E. Vigorito, S. Clare, M. V. Warren, P. Couttet, D. R. Soond, S. van Dongen, R. J. Grocock, P. P. Das, E. A. Miska, D. Vetrie, K. Okkenhaug, A. J. Enright, G. Dougan, M. Turner and A. Bradley. Requirement of bic/microRNA-155 for normal immune function. Science. 2007. 316, 608-611.
Saxena, R. and J. Kaur. Th1/Th2 cytokines and their genotypes as predictors of hepatitis B virus related hepatocellular carcinoma. World J Hepatol. 2015. 7, 1572-1580.
Seddiki, N., V. Brezar, N. Ruffin, Y. Levy and S. Swaminathan. Role of miR-155 in the regulation of lymphocyte immune function and disease. Immunology. 2014. 142, 32-38.
Sengun, A., R. Karadag, A. Karakurt, M. S. Saricaoglu, O. Abdik and H. Hasiripi. Causes of uveitis in a referral hospital in Ankara, Turkey. Ocul Immunol Inflamm. 2005. 13, 45-50.
Shao, H., D. Sun, S. L. Sun, J. M. Cruze, N. Bora and H. J. Kaplan. Expression of B7 molecules in the eye during experimental autoimmune anterior uveitis (EAAU). Curr Eye Res. 2002. 25, 271-277.
Singh, R., V. Gupta and A. Gupta. Pattern of uveitis in a referral eye clinic in north India. Indian J Ophthalmol. 2004. 52, 121-125.
Singh, S., G. Rai and A. Aggarwal. Association of microRNA-146a and its target gene IRAK1 polymorphism with enthesitis related arthritis category of juvenile idiopathic arthritis. Rheumatol Int. 2014. 34, 1395-1400.
Song, L., L. Liu, Z. Wu, Y. Li, Z. Ying, C. Lin, J. Wu, B. Hu, S. Y. Cheng, M. Li and J. Li. TGF-beta induces miR-182 to sustain NF-kappaB activation in glioma subsets. J Clin Invest. 2012. 122, 3563-3578.
Stittrich, A. B., C. Haftmann, E. Sgouroudis, A. A. Kuhl, A. N. Hegazy, I. Panse, R. Riedel, M. Flossdorf, J. Dong, F. Fuhrmann, G. A. Heinz, Z. Fang, N. Li, U. Bissels, F. Hatam, A. Jahn, B. Hammoud, M. Matz, F. M. Schulze, R. Baumgrass, A. Bosio, H. J. Mollenkopf, J. Grun, A. Thiel, W. Chen, T. Hofer, C. Loddenkemper, M. Lohning, H. D. Chang, N. Rajewsky, A. Radbruch and M. F. Mashreghi. The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes. Nat Immunol. 2010. 11, 1057-1062.
Sugita, S., Y. Kawazoe, A. Imai, Y. Yamada, S. Horie and M. Mochizuki. Inhibition of Th17 differentiation by anti-TNF-alpha therapy in uveitis patients with Behcet''s disease. Arthritis Res Ther. 2012. 14, R99.
Sun, D., D. Liang, H. J. Kaplan and H. Shao. The role of Th17-associated cytokines in the pathogenesis of experimental autoimmune uveitis (EAU). Cytokine. 2015.
Taganov, K. D., M. P. Boldin, K. J. Chang and D. Baltimore. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci U S A. 2006. 103, 12481-12486.
Tang, B., B. Xiao, Z. Liu, N. Li, E. D. Zhu, B. S. Li, Q. H. Xie, Y. Zhuang, Q. M. Zou and X. H. Mao. Identification of MyD88 as a novel target of miR-155, involved in negative regulation of Helicobacter pylori-induced inflammation. FEBS Lett. 2010. 584, 1481-1486.
Thamilarasan, M., D. Koczan, M. Hecker, B. Paap and U. K. Zettl. MicroRNAs in multiple sclerosis and experimental autoimmune encephalomyelitis. Autoimmun Rev. 2012. 11, 174-179.
Vigorito, E., K. L. Perks, C. Abreu-Goodger, S. Bunting, Z. Xiang, S. Kohlhaas, P. P. Das, E. A. Miska, A. Rodriguez, A. Bradley, K. G. Smith, C. Rada, A. J. Enright, K. M. Toellner, I. C. Maclennan and M. Turner. microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells. Immunity. 2007. 27, 847-859.
Wei, L., Q. Zhou, S. Hou, L. Bai, Y. Liu, J. Qi, Q. Xiang, Y. Zhou, A. Kijlstra and P. Yang. MicroRNA-146a and Ets-1 gene polymorphisms are associated with pediatric uveitis. PLoS One. 2014. 9, e91199.
Woon, M. D., H. J. Kaplan and N. S. Bora. Kinetics of cytokine production in experimental autoimmune anterior uveitis (EAAU). Curr Eye Res. 1998. 17, 955-961.
Wu, X., W. Hou, S. Sun, E. Bi, Y. Wang, M. Shi, J. Zang, C. Dong and B. Sun. Novel function of IFN-gamma: negative regulation of dendritic cell migration and T cell priming. J Immunol. 2006. 177, 934-943.
Xiao, C. and K. Rajewsky. MicroRNA control in the immune system: basic principles. Cell. 2009. 136, 26-36.
Yang, C. H., I. M. Fang, C. P. Lin, C. M. Yang and M. S. Chen. Effects of the NF-kappaB inhibitor pyrrolidine dithiocarbamate on experimentally induced autoimmune anterior uveitis. Invest Ophthalmol Vis Sci. 2005. 46, 1339-1347.
Yang, L. and E. Seki. Toll-like receptors in liver fibrosis: cellular crosstalk and mechanisms. Front Physiol. 2012. 3, 138.
Yenmis, G., T. Oner, C. Cam, A. Koc, O. S. Kucuk, M. C. Yakicier, D. Dizman and G. Kanigur Sultuybek. Association of NFKB1 and NFKBIA polymorphisms in relation to susceptibility of Behcet''s disease. Scand J Immunol. 2015. 81, 81-86.
Yu, H., Y. Liu, L. Bai, A. Kijlstra and P. Yang. Predisposition to Behcet''s disease and VKH syndrome by genetic variants of miR-182. J Mol Med (Berl). 2014. 92, 961-967.
Zhao, R., H. Zhou and S. B. Su. A critical role for interleukin-1beta in the progression of autoimmune diseases. Int Immunopharmacol. 2013. 17, 658-669.
Zhao, R., H. Zhou, J. Zhang, X. Liu and S. B. Su. Interleukin-1beta promotes the induction of retinal autoimmune disease. Int Immunopharmacol. 2014. 22, 285-292.
Zhou, Q., S. Hou, L. Liang, X. Li, X. Tan, L. Wei, B. Lei, A. Kijlstra and P. Yang. MicroRNA-146a and Ets-1 gene polymorphisms in ocular Behcet''s disease and Vogt-Koyanagi-Harada syndrome. Ann Rheum Dis. 2014. 73, 170-176.
Zhou, Q., X. Xiao, C. Wang, X. Zhang, F. Li, Y. Zhou, A. Kijlstra and P. Yang. Decreased microRNA-155 expression in ocular Behcet''s disease but not in Vogt Koyanagi Harada syndrome. Invest Ophthalmol Vis Sci. 2012. 53, 5665-5674.
Zhou, R., S. P. O''Hara and X. M. Chen. MicroRNA regulation of innate immune responses in epithelial cells. Cell Mol Immunol. 2011. 8, 371-379.